The overall goal is to maximize the potential of retrovirus-mediated gene transfer to airway epithelium and other in vivo targets. To do this, it is proposed to develop a new retroviral gene transfer system, based upon equine infectious anemia virus (EVIAV). Like other lentivirus members of the retrovirus family, EIAV is capable of gene transfer to non-dividing cells. This proposal is focused on the following aspects of EIAV gene transfer vector development. (i) defining the encapsidation requirements of EIAV RNA to generate safe and efficient lentiviral packaging cells; (ii) maximizing the safety and expression of EIAV RNA to generate safe and efficient lentiviral packaging cells; (ii) maximizing the safety and expression of EIAV vectors in vector producing cells; and (iii) investigating the entry and persistence of expression in human models of well differentiated airway epithelia. The focus on EIAV RNA encapsidation includes identifying important cis- acting sequence elements important for assembling vector into virus particles. Sequences important for encapsidation will be removed from expression cassettes. The focus on expression of EIAV vectors in producer cells includes determining the role of the viral regulatory/accessory genes on producing vectors in heterologous cells. In addition the effect of modifying the vector to prevent synthesis of viral related proteins and the effect of disabling the viral transcription regulatory elements will be determined. The focus on entry includes experiments designed to expand the host range of lentiviral vectors to include the apical membrane of polarized epithelia. In addition the persistence of EIAV vector of EIAV vector expression be studied in well differentiated. Our overall hypothesis is that a safe retroviral system of efficiently delivering genes of quiescent cells will have broad application for in vivo and ex vivo gene transfer to treat diseases of the lungs and other organs.